Since 1997, highly pathogenic avian influenza viruses of the H5N1 subtype have infected humans with high case fatality rates, although no sustained human-to-human transmission has yet been reported. Currently, the molecular features and mechanisms that would result in human-to-human transmission of H5N1 viruses are not understood. Attempts to select transmissible H5 viruses (which typically do not transmit among mammals) have not been successful, suggesting that influenza virus transmissibility is determined by several currently not fully understood factors. In our pilot study, we screened H5 virus libraries possessing random mutations in the globular head region of the hemagglutinin (HA) protein and identified mutant H5 HAs that acquired the ability to bind to human-type receptors. These mutant H5 viruses did not transmit among ferrets (an established influenza virus transmission model) via respiratory droplets (aerosols), but acquired this ability after two passages in ferrets that resulted in the selection of additional mutations in HA. This marks the first conversion of an H5 virus that does not transmit among ferrets into one with efficient aerosol transmission. Based on this finding, we propose to decipher the determinants of H5 virus transmission in mammals. In Aim 1, we plan To Identify and Mechanistically Characterize Amino Acid Changes in HA that Render H5 Viruses Transmissible in Mammals. To gain a better picture of mutations in HA that result in transmissible viruses, we plan to select transmissible viruses based on H5 HA proteins derived from different subclades that have caused human infections. Mutations in HA that are critical for the generation of transmissible H5 viruses will then be characterized for their receptor-binding specificity, their structural consequences, their effects in other genetic backgrounds, and their pathogenicity in mice and ferrets. We will also assess the mechanism(s) by which these mutations affect transmissibility. Mutations in HA that allow avian influenza viruses to bind to human-type receptors are most likely a critical precondition for transmission among mammals; however, findings by us and others indicate that human-type receptor binding is not sufficient for respiratory droplet transmission among ferrets, and that other viral genes also contribute to transmissibility. To Characterize the Contribution of Viral Genes Other than HA to H5 Virus Transmissibility (Aim 2), we plan to replace genes of our transmissible H5 virus with their counterparts derived from a non-transmissible virus. Viruses that no longer transmit among ferrets will then be passaged in ferrets to select mutants that transmit in these animals. The selected mutations will be characterized for their biological effects, using established assays for internalization, intracellular transport, replication and transcription, assembly and budding, and interferon antagonism. Collectively, these studies are expected to generate critical information about the molecular determinants and mechanisms of H5 virus transmissibility in mammals.